Membrane-initiated estradiol signaling induces spinogenesis required for female sexual receptivity

Abstract

Estrogens have profound actions on the structure of the nervous system during development and in adulthood. One of the signature actions of estradiol is to alter the morphology of neural processes. In the hippocampus, estradiol modulates spines and cellular excitability that affect cognitive behaviors. In the hypothalamus, estradiol increases spine density in mediobasal hypothalamic nuclei that regulate reproduction. The hypothalamic arcuate nucleus (ARH), an important site for modulation of female sexual receptivity, has a sexual dimorphism in dendritic spine density that favors females. In the present study, we used both β-actin immunostaining and Golgi staining to visualize estradiol-induced changes in spine density in Long-Evans rats. Golgi impregnation was used to visualize spine shape, and then β-actin immunoreactivity was used as a semiquantitative measure of spine plasticity since actin forms the core of dendritic spines. At 4 h after estradiol treatment, both β-actin immunofluorescence and filopodial spines were increased (from 70.57 ± 1.09% to 78.01 ± 1.05%, p < 0.05). Disruption of estradiol-induced β-actin polymerization with cytochalasin D attenuated lordosis behavior, indicating the importance of estradiol-mediated spinogenesis for female sexual receptivity (81.43 ± 7.05 to 35.00 ± 11.76, p < 0.05). Deactivation of cofilin, an actin depolymerizing factor is required for spinogenesis. Membrane-initiated estradiol signaling involving the metabotropic glutamate receptor 1a was responsible for the phosphorylation and thereby deactivation of cofilin. These data demonstrate that estradiol-induced spinogenesis in the ARH is an important cellular mechanism for the regulation of female sexual behavior.

Figure 1.

Estradiol increases spine density and maturity. A, Ovx animals treated for 4 h with EB show an increased spine density compared to oil-treated controls. At later time points the density remains elevated (n = 4–6). B, Four hours of EB treatment significantly increases the number of spines with a filopodial appearance in the ARH compared to oil-treated animals, as well as all other EB time points. Spine morphology matured at later time points after estradiol treatment (i.e., 20, 30, and 48 h). Significantly more mushroom spines were present at time points later than 4 h or in the oil-treated controls (n = 4–6). C–E, An oil-treated animal (C), an animal treated with EB for 4 h (D), and an animal treated with EB for 48 h (E). White-filled arrowheads indicate filopodial spines, and black arrowheads indicate mushroom-shaped spines. Insets show close ups of filopodial (in D) and mushroom (in E) shaped spines. Since cup-shaped and stubby spines accounted for <5% of the population of spines in the ARH, they were not included in the figure. Scale bar measures 20 μm. Error bars represent the SEM. *p < 0.05. **p < 0.001. ***p < 0.0001.

Figure 2.

CD inhibits β-actin polymerization. Each female was given three cycles of 5 μg of EB and perfused 4 h after their final dose. Before either the second or third cycle, some animals received a 400 nmol infusion of the β-actin polymerization inhibitor CD into the lateral ventricle. Animals given CD on the second cycle were able to recover the ability to make new spines by the third cycle and showed an increase in spinogenesis equal to those that had received three doses of EB only. In contrast, CD given on the third cycle, before the final dose of EB had significantly reduced levels of β-actin optical density indicating decreased spinogenesis (n = 5–7). Error bars represent the SEM. **p < 0.001.

Figure 3.

Spinogenesis is important for female sexual behavior. Ovx animals were given 5 μg of EB every 4 d for three cycles. Thirty minutes before the final estradiol treatment, females had 50 nmol of CD microinfused into the ARH. Thirty hours later, they were tested for sexual receptivity. Those that were given CD and EB had a significantly reduced LQ compared to DMSO + EB controls (n = 4–7). Error bars represent the SEM. *p < 0.01.

Figure 4.

Cofilin is deactivated by estrogen receptor interaction with mGluR1a. A, A Western blot showing a single band for p-cofilin migrating at an apparent molecular weight of ∼19 kDa. B, Ovx animals were injected with 5 μg of EB or oil and perfused 1 h later. Animals that received the mGluR1 antagonist, LY367,385, were infused 1 h before injection. C, D, There is significantly less p-cofilin in the oil- (C) than in the EB- (D) treated animals. This increase was blocked by inhibition of mGluR1, suggesting that the interaction between ERα and mGluR1 is important for the initiation of spinogenesis (n = 5–7). The ARH is outlined by the dotted line. Scale bar measures 100 μm. Error bars represent the SEM. *p < 0.01. 3V, Third ventricle.